Nucleic acid and amino acid sequences for ATP-binding cassette transporter and methods of screening for agents that modify ATP-binding cassette transporter

ABSTRACT

The present invention provides nucleic acid and amino acid sequences of an ATP binding cassette transporter and mutated sequences thereof associated with macular degeneration. Methods of detecting agents that modify ATP-binding cassette transporter comprising combining purified ATP binding cassette transporter and at least one agent suspected of modifying the ATP binding cassette transporter an observing a change in at least one characteristic associated with ATP binding cassette transporter. Methods of detecting macular degeneration is also embodied by the present invention.

[0001] This application claims priority to U.S. provisional applicationserial No. 60/039,388, filed Feb. 27, 1997.

BACKGROUND OF THE INVENTION

[0002] Macular degeneration affects approximately 1.7 millionindividuals in the U.S. and is the most common cause of acquired visualimpairment in those over the age of 65. Stargardt disease (STGD;McKusick Mendelian Inheritance (MIM) #248200) is arguably the mostcommon hereditary recessive macular dystrophy and is characterized byjuvenile to young adult onset, central visual impairment, progressivebilateral atrophy of the macular retinal pigment epithelium (RPE) andneuroepithelium, and the frequent appearance of orange-yellow flecksdistributed around the macula and/or the midretinal periphery(Stargardt, 1909; Anderson et al., 1995). A clinically similar retinaldisorder (Fundus Flavimaculatus, FFM, Franceschetti, 1963) oftendisplays later age of onset and slower progression (Fishman, 1976; Nobleand Carr, 1979). From linkage analysis, it has been concluded that STGDand FFM are most likely allelic autosomal recessive disorders withslightly different clinical manifestations caused by mutation(s) of agene at chromosome 1 p13-p21 (Gerber et al., 1995; Anderson et al.,1995). The STGD gene has been localized to a 4 cM region flanked by therecombinant markers D1S435 and D1S236 and a complete yeast artificialchromosome (YAC) contig of the region has been constructed (Anderson etal., 1995). Recently, the location of the STGD/FFM locus on humanchromosome 1p has been refined to a 2 cM interval between polymorphicmarkers D1S406 and D1S236 by genetic linkage analysis in an independentset of STGD families (Hoyng et al., 1996). Autosomal dominant disorderswith somewhat similar clinical phenotypes to STGD, identified in singlelarge North American pedigrees, have been mapped to chromosome 13q34(STGD2; MIM #153900; Zhang et al., 1994) and to chromosome 6q11-q14(STGD3; MIM #600110; Stone et al., 1994), although these conditions arenot characterized by the pathognomonic dark choroid observed byfluorescein angiography (Gass, 1987).

[0003] Members of the superfamily of mammalian ATP binding cassette(ABC) transporters are being considered as possible candidates for humandisease phenotypes. The ABC superfamily includes genes whose productsare transmembrane proteins involved in energy-dependent transport of awide spectrum of substrates across membranes (Childs and Ling, 1994;Dean and Allikmets, 1995). Many disease-causing members of thissuperfamily result in defects in the transport of specific substrates(CFTR, Riordan et al., 1989; ALD, Mosser et al., 1993; SUR, Thomas etal., 1995; PMP70, Shimozawa et al., 1992; TAP2, de la Salle et al.,1994). In eukaryotes, ABC genes encode typically four domains thatinclude two conserved ATP-binding domains (ATP) and two domains withmultiple transmembrane (TM) segments (Hyde et al. 1996). The ATP-bindingdomains of ABC genes contain motifs of characteristic conserved residues(Walker A and B motifs) spaced by 90-120 amino acids. Both thisconserved spacing and the “Signature” or “C” motif just upstream of theWalker B site distinguish members of the ABC superfamily from otherATP-binding proteins (Hyde et al., 1990; Michaelis and Berkower, 1995).These features have allowed the isolation of new ABC genes byhybridization, degenerate PCR, and inspection of DNA sequence databases(Allikmets et al., 1993, 1995; Dean et al., 1994; Luciani et al., 1994).

[0004] The characterization of twenty-one new members of the ABCsuperfamily may permit characterization and functions assigned to thesegenes by determining their map locations and their patterns ofexpression (Allikmets et al., 1996). That many known ABC genes areinvolved in inherited human diseases suggests that some of these newloci will also encode proteins mutated in specific genetic disorders.Despite regionally localizing a gene by mapping, the determination ofthe precise localization and sequence of one gene nonetheless requireschoosing the certain gene from about 250 genes, four to about fivemillion base pairs, from within the regionally localized chromosomalsite.

[0005] While advancements have been made as described above, mutationsin retina-specific ABC transporter (ABCR) in patients with recessivemacular dystrophy STGD/FFM have not yet been identified to Applicant'sknowledge. That ABCR expression is limited to photoreceptors, asdetermined by the present invention, provides evidence as to why ABCRhas not yet been sequenced. Further, the ABC1 subfamily of ABCtransporters is not represented by any homolog in yeast (Michaelis andBerkower, 1995), suggesting that these genes evolved to performspecialized functions in multicellular organisms, which also lendssupport to why the ABCR gene has been difficult to identify. Unlike ABCgenes in bacteria, the homologous genes in higher eukaryotes are muchless well studied. The fact that prokaryotes contain a large number ofABC genes suggests that many mammalian members of the superfamily remainuncharacterized. The task of studying eukaryote ABC genes is moredifficult because of the significantly higher complexity of eukaryoticsystems and the apparent difference in function of even highlyhomologous genes. While ABC proteins are the principal transporters of anumber of diverse compounds in bacterial cells, in contrast, eukaryoteshave evolved other mechanisms for the transport of many amino acids andsugars. Eukaryotes have other reasons to diversify the role of ABCgenes, for example, performing such functions as ion transport, toxinelimination, and secretion of signaling molecules.

[0006] Accordingly, there remains a need for the identification of thesequence of the gene, which in mutated forms is associated with retinaland/or macular degenerative diseases, including Stargardt Disease andFundus Flavimaculatus, for example, in order to provide enhanceddiagnoses and improved prognoses and interventional therapies forindividuals affected with such diseases.

SUMMARY OF THE INVENTION

[0007] The present invention provides sequences encoding an ATP bindingcassette transporter. Nucleic acid sequences, including SEQ ID NO: 1which is a genomic sequence, and SEQ ID NOS: 2 and 5 which are cDNAsequences, are sequences to which the present invention is directed.

[0008] A further aspect of the present invention provides ATP bindingcassette transporter polypeptides and/or proteins. SEQ ID NOS: 3 and 6are novel polypeptides of the invention produced from nucleotidesequences encoding the ATP binding cassette transporter. Also within thescope of the present invention is a purified ATP binding cassettetransporter.

[0009] The present invention also provides an expression vectorcomprising a nucleic acid sequence encoding an ATP binding cassettetransporter, a transformed host cell capable of expressing a nucleicacid sequence encoding an ATP binding cassette transporter, a cellculture capable of expressing an ATP binding cassette transporter, and aprotein preparation comprising an ATP binding cassette transporter.

[0010] The present invention is also directed to a method of screeningfor an agent that modifies ATP binding cassette transporter comprisingcombining purified ATP binding cassette transporter with an agentsuspected of modifying ATP binding cassette transporter and observing achange in at least one characteristic associated with ATP bindingcassette transporter. The present invention provides methods ofidentifying an agent that inhibits macular degeneration comprisingcombining purified ATP binding cassette transporter from a patientsuspected of having macular degeneration and an agent suspectedinteracting with the ATP binding cassette transporter and observing aninhibition in at least one of the characteristics of diseases associatedwith the ATP binding cassette transporter. In addition, the presentinvention provides for methods of identifying an agent that inducesonset of at least one characteristic associated with ATP bindingcassette transporter comprising combining purified wild-type ATP bindingcassette transporter with an agent suspected of inducing a maculardegenerative disease and observing the onset of a characteristicassociated with macular degeneration.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1A and 1B displays the ABCR gene and amplification products.FIG. 1A displays a physical map of the ABCR gene. Mega-YAC clones fromthe CEPH mega-YAC genomic library (Bellane-Chantelot et al., 1992)encompassing the 4cM critical region for STGD are represented byhorizontal bars with shaded circles indicating confirmed positives forSTSs by landmark mapping. The individual STS markers and their physicalorder are shown below the YACs with arrows indicating the centromeric(cen) and telomeric (1 pter) direction (Anderson et al., 1995). Thehorizontal double head arrow labeled STGD indicates the refined geneticinterval delineated by historical recombinants (Anderson et al., 1995).FIG. 1B displays the results of agarose gel electrophoresis of PCRamplification products with primers from the 5′ (GGTCTTCGTGTGTGGTCATT,SEQ ID NO: 114, GGTCCAGTTCTTCCAGAG, SEQ ID NO: 115, labeled 5′ ABCR) or3′ (ATCCTCTGACTCAGCAATCACA, SEQ ID NO: 116, TTGCAATTACAAATGCAATGG, SEQID NO: 117, labeled 3′ ABCR) regions of ABCR on the 13 different YAC DNAtemplates indicated as diagonals above the gel. The asterisk denotesthat YAC 680_b_(—)5 was positive for the 5′ ABCR PCR but negative forthe 3′ ABCR PCR. These data suggest the ABCR gene maps within theinterval delineated by markers D1S3361-D1S236 and is transcribed towardthe telomere, as depicted by the open horizontal box.

[0012]FIG. 2 exhibits the size and tissue distribution of ABCRtranscripts in the adult rat. A blot of total RNA from the indicatedtissues was hybridized with a 1.6 kb mouse Abcr probe (top) and aribosomal protein S26 probe (bottom; Kuwano et al., 1985). The ABCRprobe revealed a predominant transcript of approximately 8 kb that isfound in retina only. The mobility of the 28S and 18S ribosomal RNAs areindicated at the right. B, brain; H, heart; K, kidney; Li, liver; Lu,lung; R, retina; S, spleen.

[0013]FIG. 3 shows the sequence of the ABCR coding region within thegenomic ABCR sequence, SEQ ID NO: 1. The sequence of the ABCR cDNA, SEQID NO: 2, is shown with the predicted protein sequence, SEQ ID NO: 3, inone-letter amino acid code below. The location of splice sites is shownby the symbol |.

[0014]FIG. 4 displays the alignment of the ABCR protein, SEQ ID NO: 3,with other members of the ABC I subfamily. The deduced amino acidsequence of ABCR is shown aligned to known human and mouse proteins thatare members of the same subfamily. Abc1, mouse Abc1, Abc2, mouse Abc2,and ABCC, human ABC gene. The Walker A and B motifs and the Signaturemotif C are designated by underlining and the letters A, B, and C,respectively.

[0015]FIG. 5 exhibits the location of Abcr from a Jackson BSS Backcrossshowing a portion of mouse chromosome 3. The map is depicted with thecentromere toward the top. A 3 cM scale bar is also shown. Loci mappingto the same position are listed in alphabetical order.

[0016]FIG. 6 shows the segregation of SSCP variants in exon 49 of theABCR gene in kindred AR293. Sequence analysis of SSCP bands revealed theexistence of wild-type sequence (bands 1 and 3) and mutant sequence(bands 2 and 4). DNA sequencing revealed a 15 base pair deletion, whilethe affected children (lanes 2 and 3) are homozygous. Haplotype analysisdemonstrated homozygosity at the STGD locus in the two affectedindividuals.

[0017]FIG. 7A-H shows the localization of ABCR transcripts tophotoreceptor cells. In situ hybridization was performed withdigoxygenin-labeled riboprobes and visualized using an alkalinephosphatase conjugated anti-digoxygenin antibody. FIG. 7A-D displayshybridization results of retina and choroid from a pigmented mouse(C57/B16); FIG. 7E and 7F shows hybridization results of retina andchoroid from an albino rat; and FIG. 7G and 7H exhibits hybridizationresults of retina from a macaque monkey. FIG. 7A, 7E, and 7G displayresults from a mouse abcr antisense probe; FIG. 7B exhibit results froma mouse abcr sense probe; FIG. 7C shows results from a macaque rhodopsinantisense probe; and FIG. 7D, 7F, and 7H display results from a mouseblue cone pigment antisense probe. ABCR transcripts are localized to theinner segments of the photoreceptor cell layer, a pattern that matchesthe distribution of rhodopsin transcripts but is distinct from thedistribution of cone visual pigment transcripts. Hybridization is notobserved in the RPE or choroid, as seen most clearly in the albino rateye (arrowhead in FIG. 7E). The retinal layers indicated in FIG. 7B are:OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL,outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiformlayer; GCL, ganglion cell layer.

[0018]FIG. 8 provides a pGEM®-T Vector map.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention is directed to the nucleic acid and proteinsequences encoding ATP binding cassette transporter. The ATP bindingcassette transporter of the present invention is retina specific ATPbinding cassette transporter (ABCR); more particularly, ABCR may beisolated from retinal cells, preferably photoreceptor cells. The presentinvention provides nucleotide sequences of ABCR including genomicsequences, SEQ ID NO: 1, and cDNA sequences SEQ ID NO: 2 and 5. Novelpolypeptide sequences, SEQ ID NOS: 3 and 6, for ABCR, are the translatedproducts of SEQ ID NOS: 2 and 5, respectively, and are also included inthe present invention.

[0020] SEQ ID NO: 1 provides the human genomic DNA sequence of ABCR. SEQID NOS: 2 and 5 provide wild-type cDNA sequences of human ABCR, whichresult in translated products SEQ ID NOS: 3 and 6, respectively. Whilenot intending to be bound by any particular theory or theories ofoperation, it is believed that SEQ ID NOS: 2 and 5 are isoforms of ABCRcDNA. The difference between SEQ ID NOS: 2 and 5 may be accounted for byan additional sequence in SEQ ID NO: 2 which is added between bases 4352and 4353 of SEQ ID NO: 5. This difference is thought to arise fromalternative splicing of the nascent transcript of ABCR, in which analternative exon 30, SEQ ID NO: 4, is excluded. This alternative exonencodes an additional 38 amino acids, SEQ ID NO: 11.

[0021] Nucleic acids within in the scope of the present inventioninclude cDNA, RNA, genomic DNA, fragments or portions within thesequences, antisense oligonucleotides. Sequences encoding the ABCR alsoinclude amino acid, polypeptide, and protein sequences. Variations inthe nucleic acid and polypeptide sequences of the present invention arewithin the scope of the present invention and include N terminal and Cterminal extensions, transcription and translation modifications, andmodifications in the cDNA sequence to facilitate and improvetranscription and translation efficiency. In addition, changes withinthe wild-type sequences identified herein which changed sequence retainssubstantially the same wild-type activity, such that the changedsequences are substantially similar to the ABCR sequences identified,are also considered within the scope of the present invention.Mismatches, insertions, and deletions which permit substantialsimilarity to the ABCR sequences, such as similarity in residues inhydrophobicity, hydrophilicity, basicity, and acidity, will be known tothose of skill in the art once armed with the present disclosure. Inaddition, the isolated, or purified, sequences of the present inventionmay be natural, recombinant, synthetic, or a combination thereof.Wild-type activity associated with the ABCR sequences of the presentinvention include, inter alia, all or part of a sequence, or a sequencesubstantially similar thereto, that codes for ATP binding cassettetransporter.

[0022] The genomic, SEQ ID NO: 1, and cDNA, SEQ ID NOS: 2 and 5,sequences are identified in FIG. 3 and encode ABCR, certain mutations ofwhich are responsible for the class of retinal disorders known asretinal or macular degenerations. Macular degeneration is characterizedby macular dystrophy, various alterations of the peripheral retina,central visual impairment, progressive bilateral atrophy of the macularretinal pigment epithelium (RPE) and neuroepithelium, frequentappearance of orange-yellow flecks distributed around the macula and/orthe midretinal periphery, and subretinal deposition of lipofuscin-likematerial. Retinal and macular degenerative diseases include and are notlimited to Stargardt Disease, Fundus Flavimaculatus, age-related maculardegeneration, and may include disorders variously called retinitispigmentosa, combined rod and cone dystrophies, cone dystrophies anddegenerations, pattern dystrophy, bull's eye maculopathies, and variousother retinal degenerative disorders, some induced by drugs, toxins,environmental influences, and the like. Stargardt Disease is anautosomal recessive retinal disorder characterized by juvenile toadult-onset macular and retinal dystrophy. Fundus Flavimaculatus oftendisplays later age of onset and slower progression. Some environmentalinsults and drug toxicities may create similar retinal degenerations.Linkage analysis reveals that Stargardt Disease and FundusFlavimaculatus may be allelic autosomal recessive disorders withslightly different clinical manifestations. The identification of theABCR gene suggests that different mutations within ABCR may beresponsible for these clinical phenomena.

[0023] The present invention is also directed to a method of screeningfor an agent that modifies ATP binding cassette transporter comprisingcombining purified ATP binding cassette transporter with an agentsuspected of modifying ATP binding cassette transporter and observing achange in at least one characteristic associated with ATP bindingcassette transporter.

[0024] “Modify” and variations thereof include changes such as and notlimited to inhibit, suppress, delay, retard, slow, suspend, obstruct,and restrict, as well as induce, encourage, provoke, and cause. Modifymay also be defined as complete inhibition such that maculardegeneration is arrested, stopped, or blocked. Modifications may,directly or indirectly, inhibit or substantially inhibit, maculardegeneration or induce, or substantially induce, macular degeneration,under certain circumstances.

[0025] Methods of identifying an agent that inhibits maculardegeneration are embodied by the present invention and comprisecombining purified ATP binding cassette transporter from a patientsuspected of having macular degeneration and an agent suspected ofinteracting with the ATP binding cassette transporter and observing aninhibition in at least one of the characteristics of diseases associatedwith the ATP binding cassette transporter. Accordingly, such methodsserve to reduce or prevent macular degeneration, such as in humanpatients. In addition, the present invention provides for methods ofidentifying an agent that induces onset of at least one characteristicassociated with ATP binding cassette transporter comprising combiningpurified wild-type ATP binding cassette transporter with an agentsuspected of inducing a macular degenerative disease and observing theonset of a characteristic associated with macular degeneration. Thus,such methods provide methods of using laboratory animals to determinecausative agents of macular degeneration. The ATP binding cassettetransporter may be provided for in the methods identified herein in theform of nucleic acids, such as and not limited to SEQ ID NOS: 1, 2, and5 or as an amino acid, SEQ ID NOS: 3 and 6, for example. Accordingly,transcription and translation inhibitors may be separately identified.Characteristics associated with macular degeneration include and are notlimited to central visual impairment, progressive bilateral atrophy ofthe macular retinal pigment epithelium (RPE) and neuroepithelium, andthe frequent appearance of orange-yellow flecks distributed around themacula and/or the midretinal periphery. Accordingly, observing one ormore of the characteristics set forth above results in identification ofan agent that induces macular degeneration, whereas reduction orinhibition of at least one of the characteristics results inidentification of an agent that inhibits macular degeneration.

[0026] Mutational analysis of ABCR in Stargardt Disease familiesrevealed thus far seventy four mutations including fifty four singleamino acid substitutions, five nonsense mutations resulting in earlytruncation of the protein, six frame shift mutations resulting in earlytruncation of the protein, three in-frame deletions resulting in loss ofamino acid residues from the protein, and six splice site mutationsresulting in incorrect processing of the nascent RNA transcript, seeTable 2. Compound heterozygotes for mutations in ABCR were found inforty two families. Homozygous mutations were identified in threefamilies with consanguineous parentage. Accordingly, mutations inwild-type ABCR which result in activities that are not associated withwild-type ABCR are herein referred to as sequences which are associatedwith macular degeneration. Such mutations include missense mutations,deletions, insertions, substantial differences in hydrophobicity,hydrophilicity, acidity, and basicity. Characteristics which areassociated with retinal or macular degeneration include and are notlimited to those characteristics set forth above.

[0027] Mutations in wild-type ABCR provide a method of detecting maculardegeneration. Retinal or macular degeneration may be detected byobtaining a sample comprising patient nucleic acids from a patienttissue sample; amplifying retina-specific ATP binding cassette receptorspecific nucleic acids from the patient nucleic acids to produce a testfragment; obtaining a sample comprising control nucleic acids from acontrol tissue sample; amplifying control nucleic acids encodingwild-type retina-specific ATP binding cassette receptor to produce acontrol fragment; comparing the test fragment with the control fragmentto detect the presence of a sequence difference in the test fragment,wherein a difference in the test fragment indicates maculardegeneration. Mutations in the test fragment, including and not limitedto each of the mutations identified above, may provide evidence ofmacular degeneration.

[0028] A purified ABCR protein is also provided by the presentinvention. The purified ABCR protein may have an amino acid sequence asprovided by SEQ ID NOS: 3 and 6.

[0029] The present invention is directed to ABCR sequences obtained frommammals from the Order Rodentia, including and not limited to hamsters,rats, and mice; Order Logomorpha, such as rabbits; more particularly theOrder Carnivora, including Felines (cats) and Canines (dogs); even moreparticularly the Order Artiodactyla, Bovines (cows) and Suines (pigs);and the Order Perissodactyla, including Equines (horses); and mostparticularly the Order Primates, Ceboids and Simoids (monkeys) andAnthropoids (humans and apes). The mammals of most preferred embodimentsare humans.

[0030] Generally, the sequences of the invention may be produced in hostcells transformed with an expression vector comprising a nucleic acidsequence encoding ABCR. The transformed cells are cultured underconditions whereby the nucleic acid sequence coding for ABCR isexpressed. After a suitable amount of time for the protein toaccumulate, the protein may be purified from the transformed cells.

[0031] A gene coding for ABCR may be obtained from a cDNA library.Suitable libraries can be obtained from commercial sources such asClontech, Palo Alto, Calif. Libraries may also be prepared using thefollowing non-limiting examples: hamster insulin-secreting tumor (HIT),mouse αTC-6, and rat insulinoma (RIN) cells. Positive clones are thensubjected to DNA sequencing to determine the presence of a DNA sequencecoding for ABCR. DNA sequencing is accomplished using the chaintermination method of Sanger et al., Proc. Nat'l. Acad. Sci, U.S.A.,1977, 74, 5463. The DNA sequence encoding ABCR is then inserted into anexpression vector for later expression in a host cell.

[0032] Expression vectors and host cells are selected to form anexpression system capable of synthesizing ABCR. Vectors including andnot limited to baculovirus vectors may be used in the present invention.Host cells suitable for use in the invention include prokaryotic andeukaryotic cells that can be transformed to stably contain and expressABCR. For example, nucleic acids coding for the recombinant protein maybe expressed in prokaryotic or eukaryotic host cells, including the mostcommonly used bacterial host cell for the production of recombinantproteins, E. coli. Other microbial strains may also be used, however,such as Bacillus subtilis, and other enterobacteriaceae such asSalmonella typhimurium or Serratia marcescens, various species ofPseudomonas, or other bacterial strains.

[0033] The preferable eukaryotic system is yeast, such as Saccharomycescerevisiae. Yeast artificial chromosome (YAC) systems are able toaccommodate the large size of ABCR gene sequence or genomic clone. Theprinciple of the YAC system is similar to that used in conventionalcloning of DNA. Large fragments of cDNA are ligated into two “arms” of aYAC vector, and the ligation mixture is then introduced into the yeastby transformation. Each of the arms of the YAC vector carries aselectable marker as well as appropriately oriented sequences thatfunction as telomeres in yeast. In addition, one of the two arms carriestwo small fragments that function as a centromere and as an origin ofreplication (also called an ARS element-autonomously replicatingsequences). Yeast transformants that have taken up and stably maintainedan artificial chromosome are identified as colonies on agar platescontaining the components necessary for selection of one or both YACarms. YAC vectors are designed to allow rapid identification oftransformants that carry inserts of genomic DNA. Insertion of genomicDNA into the cloning site interrupts a suppressor tRNA gene and resultsin the formation of red rather than white colonies by yeast strains thatcarry an amber ade2 gene.

[0034] To clone in YAC vectors, genomic DNA from the test organism isprepared under conditions that result in relatively little shearing suchthat its average size is several million base pairs. The cDNA is thenligated to the arms of the YAC vector, which has been appropriatelyprepared to prevent self-ligation. As an alternative to partialdigestion with EcoRI, YAC vectors may be used that will accept genomicDNA that has been digested to completion with rarely cutting restrictionenzymes such as NotI or MluI.

[0035] In addition, insect cells, such as Spodoptera frugiperda; chickencells, such as E3C/O and SL-29; mammalian cells, such as HeLa, Chinesehamster ovary cells (CHO), COS-7 or MDCK cells and the like may also beused. The foregoing list is illustrative only and is not intended in anyway to limit the types of host cells suitable for expression of thenucleic acid sequences of the invention.

[0036] As used herein, expression vectors refer to any type of vectorthat can be manipulated to contain a nucleic acid sequence coding forABCR, such as plasmid expression vectors, viral vectors, and yeastexpression vectors. The selection of the expression vector is based oncompatibility with the desired host cell such that expression of thenucleic acid encoding ABCR results. Plasmid expression vectors comprisea nucleic acid sequence of the invention operably linked with at leastone expression control element such as a promoter. In general, plasmidvectors contain replicon and control sequences derived from speciescompatible with the host cell. To facilitate selection of plasmidscontaining nucleic acid sequences of the invention, plasmid vectors mayalso contain a selectable marker such as a gene coding for antibioticresistance. Suitable examples include the genes coding for ampicillin,tetracycline, chloramphenicol, or kanamycin resistance.

[0037] Suitable expression vectors, promoters, enhancers, and otherexpression control elements are known in the art and may be found inSambrook et al., Molecular Cloning: A Laboratory Manual, second edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989),incorporated herein by reference in its entirety.

[0038] Transformed host cells containing a DNA sequence encoding ABCRmay then be grown in an appropriate medium for the host. The cells arethen grown until product accumulation reaches desired levels at whichtime the cells are then harvested and the protein product purified inaccordance with conventional techniques. Suitable purification methodsinclude, but are not limited to, SDS PAGE electrophoresis,phenylboronate-agarose, reactive green 19-agarose, concanavalin Asepharose, ion exchange chromatography, affinity chromatography,electrophoresis, dialysis and other methods of purification known in theart.

[0039] Protein preparations, of purified or unpurified ABCR by hostcells, are accordingly produced which comprise ABCR and other materialsuch as host cell components and/or cell medium, depending on the degreeof purification of the protein.

[0040] The invention also includes a transgenic non-human animal,including and not limited to mammals, such as and not limited to amouse, rat, or hamster, comprising a sequence encoding ABCR, or fragmentthereof that substantially retains ABCR activity, introduced into theanimal or an ancestor of the animal. The sequence may be wild-type ormutant and may be introduced into the animal at the embryonic or adultstage. The sequence is incorporated into the genome of an animal suchthat it is chromosomally incorporated into an activated state. Atransgenic non-human animal has germ cells and somatic cells thatcontain an ABCR sequence. Embryo cells may be transfected with the geneas it occurs naturally, and transgenic animals are selected in which thegene has integrated into the chromosome at a locus which results inactivation. Other activation methods include modifying the gene or itscontrol sequences prior to introduction into the embryo. The embryo maybe transfected using a vector containing the gene.

[0041] In addition, a transgenic non-human animal may be engineeredwherein ABCR is suppressed. For purposes of the present invention,suppression of ABCR includes, and is not limited to strategies whichcause ABCR not to be expressed. Such strategies may include and are notlimited to inhibition of protein synthesis, pre-mRNA processing, or DNAreplication. Each of the above strategies may be accomplished byantisense inhibition of ABCR gene expression. Many techniques fortransferring antisense sequences into cells are known to those of skill,including and not limited to microinjection, viral-mediated transfer,somatic cell transformation, transgene integration, and the like, as setforth in Pinkert, Carl, Transgenic Animal Technology, 1994, AcademicPress, Inc., San Diego, Calif., incorporated herein by reference in itsentirety.

[0042] Further, a transgenic non-human animal may be prepared such thatABCR is knocked out. For purposes of the present invention, a knock-outincludes and is not limited to disruption or rendering null the ABCRgene. A knock-out may be accomplished, for example, with antisensesequences for ABCR. The ABCR gene may be knocked out by injection of anantisense sequence for all or part of the ABCR sequence such as anantisense sequence for all or part of SEQ ID NO: 2. Once ABCR has beenrendered null, correlation of the ABCR to macular degeneration may betested. Sequences encoding mutations affecting the ABCR may be insertedto test for alterations in various retinal and macular degenerationsexhibited by changes in the characteristics associated with retinal andmacular degeneration. ANABCR knock-out may be engineered by insertingsynthetic DNA into the animal chromosome by homologous recombination. Inthis method, sequences flanking the target and insert DNA are identical,allowing strand exchange and crossing over to occur between the targetand insert DNA. Sequences to be inserted typically include a gene for aselectable marker, such as drug resistance. Sequences to be targeted aretypically coding regions of the genome, in this case part of the ABCRgene. In this process of homologous recombination, targeted sequencesare replaced with insert sequences thus disrupting the targeted gene andrendering it nonfunctional. This nonfunctional gene is called a nullallele of the gene.

[0043] To create the knockout mouse, a DNA construct containing theinsert DNA and flanking sequences is made. This DNA construct istransfected into pluripotent embryonic stem cells competent forrecombination. The identical flanking sequences align with one another,and chromosomal recombination occurs in which the targeted sequence isreplaced with the insert sequence, as described in Bradley, A.,Production and Analysis of Chimeric Mice, in Teratocarcinomas andEmbryonic Stem Cells—A Practical Approach, 1987, E. Roberson, Editor,IRC Press, pages 113-151. The stem cells are injected into an embryo,which is then implanted into a female animal and allowed to be born. Theanimals may contain germ cells derived from the injected stem cells, andsubsequent matings may produce animals heterozygous and homozygous forthe disrupted gene.

[0044] Transgenic non-human animals may also be useful for testingnucleic acid changes to identify additional mutations responsible formacular degeneration. A transgenic non-human animal may comprise arecombinant ABCR.

[0045] The present invention is also directed to gene therapy. Forpurposes of the present invention, gene therapy refers to the transferand stable insertion of new genetic information into cells for thetherapeutic treatment of diseases or disorders. A foreign sequence orgene is transferred into a cell that proliferates to spread the newsequence or gene throughout the cell population. Sequences includeantisense sequence of all or part of ABCR, such as an antisense sequenceto all or part of the sequences identified as SEQ ID NO: 1, 2, and 5.Known methods of gene transfer include microinjection, electroporation,liposomes, chromosome transfer, transfection techniques,calcium-precipitation transfection techniques, and the like. In theinstant case, macular degeneration may result from a loss of genefunction, as a result of a mutation for example, or a gain of genefunction, as a result of an extra copy of a gene, such as three copiesof a wild-type gene, or a gene over expressed as a result of a mutationin a promoter, for example. Expression may be altered by activating ordeactivating regulatory elements, such as a promoter. A mutation may becorrected by replacing the mutated sequence with a wild-type sequence orinserting an antisense sequence to bind to an over expressed sequence orto a regulatory sequence.

[0046] Numerous techniques are known in the art for the introduction offoreign genes into cells and may be used to construct the recombinantcells for purposes of gene therapy, in accordance with this embodimentof the invention. The technique used should provide for the stabletransfer of the heterologous gene sequence to the stem cell, so that theheterologous gene sequence is heritable and expressible by stem cellprogeny, and so that the necessary development and physiologicalfunctions of the recipient cells are not disrupted. Techniques which maybe used include but are not limited to chromosome transfer (e.g., cellfusion, chromosome-mediated gene transfer, micro cell-mediated genetransfer), physical methods (e.g., transfection, spheroplast fusion,microinjection, electroporation, liposome carrier), viral vectortransfer (e.g., recombinant DNA viruses, recombinant RNA viruses) andthe like (described in Cline, M. J., 1985, Pharmac. Ther. 29:69-92,incorporated herein by reference in its entirety).

[0047] The term “purified”, when used to describe the state of nucleicacid sequences of the invention, refers to nucleic acid sequencessubstantially free of nucleic acid not coding for ABCR or othermaterials normally associated with nucleic acid in non-recombinantcells, i.e., in its “native state.”

[0048] The term “purified” or “in purified form” when used to describethe state of an ABCR nucleic acid, protein, polypeptide, or amino acidsequence, refers to sequences substantially free, to at least somedegree, of cellular material or other material normally associated withit in its native state. Preferably the sequence has a purity(homogeneity) of at least about 25% to about 100%, More preferably thepurity is at least about 50%, when purified in accordance with standardtechniques known in the art.

[0049] In accordance with methods of the present invention, methods ofdetecting retinal or macular degenerations in a patient are providedcomprising obtaining a patient tissue sample for testing. The tissuesample may be solid or liquid, a body fluid sample such as and notlimited to blood, skin, serum, saliva, sputum, mucus, bone marrow,urine, lymph, and a tear; and feces. In addition, a tissue sample fromamniotic fluid or chorion may be provided for the detection of retinalor macular degeneration in utero in accordance with the presentinvention.

[0050] A test fragment is defined herein as an amplified samplecomprising ABCR-specific nucleic acids from a patient suspected ofhaving retinal or macular degeneration. A control fragment is anamplified sample comprising normal or wild-type ABCR-specific nucleicacids from an individual not suspected of having retinal or maculardegeneration.

[0051] The method of amplifying nucleic acids may be the polymerasechain reaction using a pair of primers wherein at least one primerwithin the pair is selected from the group consisting of SEQ ID NOS:12-113. When the polymerase chain reaction is the amplification methodof choice, a pair of primers may be used such that one primer of thepair is selected from the group consisting of SEQ ID NOS: 12-113.

[0052] Nucleic acids, such as DNA (such as and not limited to genomicDNA and cDNA) and/or RNA (such as and not limited to mRNA), are obtainedfrom the patient sample. Preferably RNA is obtained.

[0053] Nucleic acid extraction is followed by amplification of the sameby any technique known in the art. The amplification step includes theuse of at least one primer sequence which is complementary to a portionof ABCR-specific expressed nucleic acids or sequences on flankingintronic genomic sequences in order to amplify exon or coding sequences.Primer sequences useful in the amplification methods include and are notlimited to SEQ ID NOS: 12-113, which may be used in the amplificationmethods. Any primer sequence of about 10 nucleotides to about 35nucleotides, more preferably about 15 nucleotides to about 30nucleotides, even more preferably about 17 nucleotides to about 25nucleotides may be useful in the amplification step of the methods ofthe present invention. In addition, mismatches within the sequencesidentified above, which achieve the methods of the invention, such thatthe mismatched sequences are substantially complementary and thushybridizable to the sequence sought to be identified, are alsoconsidered within the scope of the disclosure. Mismatches which permitsubstantial similarity to SEQ ID NOS: 12-113, such as and not limited tosequences with similar hydrophobicity, hydrophilicity, basicity, andacidity, will be known to those of skill in the art once armed with thepresent disclosure. The primers may also be unmodified or modified.Primers may be prepared by any method known in the art such as bystandard phosphoramidite chemistry. See Sambrook et al., supra.

[0054] The method of amplifying nucleic acids may be the polymerasechain reaction using a pair of primers wherein at least one primerwithin the pair is selected from the group consisting of SEQ ID NOS:12-113. When the polymerase chain reaction is the amplification methodof choice, a pair of primers may be used such that one primer of thepair is selected from the group consisting of SEQ ID NOS: 12-113.

[0055] When an amplification method includes the use of two primers, afirst primer and a second primer, such as in the polymerase chainreaction, one of the first primer or second primer may be selected fromthe group consisting of SEQ ID NOS: 12-113. Any primer pairs which copyand amplify nucleic acids between the pairs pointed toward each otherand which are specific for ABCR may be used in accordance with themethods of the present invention.

[0056] A number of template dependent processes are available to amplifythe target sequences of interest present in a sample. One of the bestknown amplification methods is the polymerase chain reaction (PCR) whichis described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159, and in Innis et al., PCR Protocols, Academic Press, Inc., SanDiego Calif., 1990, each of which is incorporated herein by reference inits entirety. Briefly, in PCR, two primer sequences are prepared whichare complementary to regions on opposite complementary strands of thetarget sequence. An excess of deoxynucleoside triphosphates are added toa reaction mixture along with a DNA polymerase (e.g., Taq polymerase).If the target sequence is present in a sample, the primers will bind tothe target and the polymerase will cause the primers to be extendedalong the target sequence by adding on nucleotides. By raising andlowering the temperature of the reaction mixture, the extended primerswill dissociate from the target to form reaction products, excessprimers will bind to the target and to the reaction products and theprocess is repeated. Alternatively, a reverse transcriptase PCRamplification procedure may be performed in order to quantify the amountof mRNA amplified. Polymerase chain reaction methodologies are wellknown in the art.

[0057] Another method for amplification is the ligase chain reaction(referred to as LCR), disclosed in EPA No.320,308, incorporated hereinby reference in its entirety. In LCR, two complementary probe pairs areprepared, and in the presence of the target sequence, each pair willbind to opposite complementary strands of the target such that theyabut. In the presence of a ligase, the two probe pairs will link to forma single unit. By temperature cycling, as in PCR, bound ligated unitsdissociate from the target and then serve as “target sequences” forligation of excess probe pairs. U.S. Pat. No. 4,883,750, incorporatedherein by reference in its entirety, describes an alternative method ofamplification similar to LCR for binding probe pairs to a targetsequence.

[0058] Qbeta Replicase, described in PCT Application No. PCT/US87/00880,incorporated herein by reference in its entirety, may also be used asstill another amplification method in the present invention. In thismethod, a replicative sequence of RNA which has a region complementaryto that of a target is added to a sample in the presence of an RNApolymerase. The polymerase will copy the replicative sequence which canthen be detected.

[0059] An isothermal amplification method, in which restrictionendonucleases and ligases are used to achieve the amplification oftarget molecules that contain nucleotide 5′-[ alpha -thio]triphosphatesin one strand of a restriction site (Walker, G. T., et al., Proc. Natl.Acad, Sci (U.S.A) 1992, 89:392-396, incorporated herein by reference inits entirety), may also be useful in the amplification of nucleic acidsin the present invention.

[0060] Strand Displacement Amplification (SDA) is another method ofcarrying out isothermal amplification of nucleic acids which involvesmultiple rounds of strand displacement and synthesis, i.e. nicktranslation. A similar method, called Repair Chain Reaction (RCR) isanother method of amplification which may be useful in the presentinvention and which involves annealing several probes throughout aregion targeted for amplification, followed by a repair reaction inwhich only two of the four bases are present. The other two bases can beadded as biotinylated derivatives for easy detection. A similar approachis used in SDA.

[0061] ABCR-specific nucleic acids can also be detected using a cyclicprobe reaction (CPR). In CPR, a probe having a 3′ and 5′ sequences ofnon-ABCR specific DNA and middle sequence of ABCR specific RNA ishybridized to DNA which is present in a sample. Upon hybridization, thereaction is treated with RNaseH, and the products of the probeidentified as distinctive products, generate a signal which is releasedafter digestion. The original template is annealed to another cyclingprobe and the reaction is repeated. Thus, CPR involves amplifying asignal generated by hybridization of a probe to a ABCR-specificexpressed nucleic acid.

[0062] Still other amplification methods described in GB Application No.2 202 328, and in PCT Application No. PCT/US89/01025, each of which isincorporated by reference in its entirety, may be used in accordancewith the present invention. In the former application, “modified”primers are used in a PCR like, template and enzyme dependent synthesis.The primers may be modified by labeling with a capture moiety (e.g.,biotin) and/or a detector moiety (e.g., enzyme). In the latterapplication, an excess of labeled probes are added to a sample. In thepresence of the target sequence, the probe binds and is cleavedcatalytically. After cleavage, the target sequence is released intact tobe bound by excess probe. Cleavage of the labeled probe signals thepresence of the target sequence.

[0063] Other nucleic acid amplification procedures includetranscription-based amplification systems (TAS) (Kwoh D., et al., Proc.Natl. Acad. Sci. (U.S.A.) 1989, 86:1173, Gingeras T. R., et al., PCTApplication WO 88/10315, each of which is incorporated herein byreference in its entirety), including nucleic acid sequence basedamplification (NASBA) and 3SR. In NASBA, the nucleic acids can beprepared for amplification by standard phenol/chloroform extraction,heat denaturation of a clinical sample, treatment with lysis buffer andminispin columns for isolation of DNA and RNA or guanidinium chlorideextraction of RNA. These amplification techniques involve annealing aprimer which has ABCR-specific sequences. Following polymerization,DNA/RNA hybrids are digested with RNase H while double stranded DNAmolecules are heat denatured again. In either case the single strandedDNA is made fully double stranded by addition of second ABCR-specificprimer, followed by polymerization. The double stranded DNA moleculesare then multiply transcribed by a polymerase such as T7 or SP6. In anisothermal cyclic reaction, the RNAs are reverse transcribed into doublestranded DNA, and transcribed once again with a polymerase such as T7 orSP6. The resulting products, whether truncated or complete, indicateABCR-specific sequences.

[0064] Davey, C., et al., European Patent Application Publication No.329,822, incorporated herein by reference in its entirety, disclose anucleic acid amplification process involving cyclically synthesizingsingle-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (“dsDNA”)which may be used in accordance with the present invention. The ssRNA isa first template for a first primer oligonucleotide, which is elongatedby reverse transcriptase (RNA-dependent DNA polymerase). The RNA is thenremoved from resulting DNA:RNA duplex by the action of ribonuclease H(RNase H, an RNase specific for RNA in a duplex with either DNA or RNA).The resultant ssDNA is a second template for a second primer, which alsoincludes the sequences of an RNA polymerase promoter (exemplified by T7RNA polymerase) 5′ to its homology to its template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting as a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

[0065] Miller, H. I., et al., PCT application WO 89/06700, incorporatedherein by reference in its entirety, disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic; i.e. new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “race” disclosed byFrohman, M. A., In: PCR Protocols: A Guide to Methods and Applications1990, Academic Press, N.Y.) and “one-sided PCR” (Ohara, O., et al.,Proc. Natl. Acad. Sci. (U.S.A) 1989, 86:5673-5677), all referencesherein incorporated by reference in their entirety.

[0066] Methods based on ligation of two (or more) oligonucleotides inthe presence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide (Wu, D.Y. et al., Genomics 1989, 4:560, incorporated herein by reference in itsentirety), may also be used in the amplification step of the presentinvention.

[0067] Test fragment and control fragment may be amplified by anyamplification methods known to those of skill in the art, including andnot limited to the amplification methods set forth above. For purposesof the present invention, amplification of sequences encoding patientand wild-type ABCR includes amplification of a portion of a sequencesuch as and not limited to a portion of an ABCR sequence of SEQ ID NO:1, such as sequence of a length of about 10 nucleotides to about 1,000nucleotides, more preferably about 10 nucleotides to about 100nucleotides, or having at least 10 nucleotides occurring anywhere withinthe SEQ ID NO: 1, where sequence differences are known to occur withinABCR test fragments. Thus, for example, a portion of the sequenceencoding ABCR of a patient sample and a control sample may be amplifiedto detect sequence differences between these two sequences.

[0068] Following amplification of the test fragment and controlfragment, comparison between the amplification products of the testfragment and control fragment is carried out. Sequence changes such asand not limited to nucleic acid transition, transversion, andrestriction digest pattern alterations may be detected by comparison ofthe test fragment with the control fragment.

[0069] Alternatively, the presence or absence of the amplificationproduct may be detected. The nucleic acids are fragmented into varyingsizes of discrete fragments. For example, DNA fragments may be separatedaccording to molecular weight by methods such as and not limited toelectrophoresis through an agarose gel matrix. The gels are thenanalyzed by Southern hybridization. Briefly, DNA in the gel istransferred to a hybridization substrate or matrix such as and notlimited to a nitrocellulose sheet and a nylon membrane. A labeled probeencoding an ABCR mutation is applied to the matrix under selectedhybridization conditions so as to hybridize with complementary DNAlocalized on the matrix. The probe may be of a length capable of forminga stable duplex. The probe may have a size range of about 200 to about10,000 nucleotides in length, preferably about 500 nucleotides inlength, and more preferably about 2,454 nucleotides in length.Mismatches which permit substantial similarity to the probe, such as andnot limited to sequences with similar hydrophobicity, hydrophilicity,basicity, and acidity, will be known to those of skill in the art oncearmed with the present disclosure. Various labels for visualization ordetection are known to those of skill in the art, such as and notlimited to fluorescent staining, ethidium bromide staining for example,avidin/biotin, radioactive labeling such as ³²P labeling, and the like.Preferably, the product, such as the PCR product, may be run on anagarose gel and visualized using a stain such as ethidium bromide. SeeSambrook et al., supra. The matrix may then be analyzed byautoradiography to locate particular fragments which hybridize to theprobe. Yet another alternative is the sequencing of the test fragmentand the control fragment to identify sequence differences. Methods ofnucleic acid sequencing are known to those of skill in the art,including and not limited to the methods of Maxam and Gilbert, Proc.Natl. Acad. Sci., USA 1977, 74, 560-564 and Sanger, Proc. Natl. Acad.Sci., USA 1977, 74, 5463-5467.

[0070] A pharmaceutical composition comprising all or part of a sequencefor ABCR may be delivered to a patient suspected of having retinal ormacular degeneration. The sequence may be an antisense sequence. Thecomposition of the present invention may be administered alone or maygenerally be administered in admixture with a pharmaceutical carrier.The pharmaceutically-acceptable carrier may be selected with regard tothe intended route of administration and the standard pharmaceuticalpractice. The dosage will be about that of the sequence alone and willbe set with regard to weight, and clinical condition of the patient. Theproportional ratio of active ingredient to carrier will naturallydepend, inter alia, on the chemical nature, solubility, and stability ofthe sequence, as well as the dosage contemplated.

[0071] The sequences of the invention may be employed in the method ofthe invention singly or in combination with other compounds, includingand not limited to other sequences set forth in the present invention.The method of the invention may also be used in conjunction with othertreatments such as and not limited to antibodies, for example. For invivo applications the amount to be administered will also depend on suchfactors as the age, weight, and clinical condition of the patient. Thecomposition of the present invention may be administered by any suitableroute, including as an eye drop, inoculation and injection, for example,intravenous, intraocular, oral, intraperitoneal, intramuscular,subcutaneous, topically, and by absorption through epithelial ormucocutaneous linings, for example, conjunctival, nasal, oral, vaginal,rectal and gastrointestinal.

[0072] The mode of administration of the composition may determine thesites in the organism to which the compound will be delivered. Forinstance, topical application may be administered in creams, ointments,gels, oils, emulsions, pastes, lotions, and the like. For parenteraladministration, the composition maybe used in the form of sterileaqueous or non-aqueous solution which may contain another solute, forexample, sufficient salts, glucose or dextrose to make the solutionisotonic. A non-aqueous solution may be comprise an oil, for example.For oral mode of administration, the present invention may be used inthe form of tablets, capsules, lozenges, troches, powders, syrups,elixirs, aqueous solutions and suspension, and the like. Variousdisintegrants, such as starch, and lubricating agents may be used. Fororal administration in capsule form, useful diluents are lactose andhigh molecular weight polyethylene glycols. When aqueous suspensions arerequired for oral use, certain sweetening and/or flavoring agents may beadded.

[0073] A diagnostic kit for detecting retinal or macular degenerationcomprising in one or more containers at least one primer which iscomplementary to an ABCR sequence and a means for visualizing amplifiedDNA is also within the scope of the present invention. Alternatively,the kit may comprise two primers. In either case, the primers may beselected from the group consisting of SEQ ID NOS: 12-113, for example.The diagnostic kit may comprise a pair of primers wherein one primerwithin said pair is complementary to a region of the ABCR gene, whereinone of said pair of primers is selected from the group consisting of SEQID NO: 12-113, a probe specific to the amplified product, and a meansfor visualizing amplified DNA, and optionally including one or more sizemarkers, and positive and negative controls. The diagnostic kit of thepresent invention may comprise one or more of a fluorescent dye such asethidium bromide stain, ³²p, and biotin, as a means for visualizing ordetecting amplified DNA. Optionally the kit may include one or more sizemarkers, positive and negative controls, restriction enzymes, and/or aprobe specific to the amplified product.

[0074] The following examples are illustrative but are not meant to belimiting of the invention.

EXAMPLES: Identification of the ABCR as a Candidate Gene for STGD

[0075] One of the 21 new human genes from the ABC superfamily, hereaftercalled ABCR (retina-specific ABC transporter), was identified (Allikmetset al. 1996) among expressed sequence tags (ESTs) obtained from 5,000human retina cDNA clones (Wang, Y., Macke, J. P., Abella, B. S.,Andreasson, K., Worley, P., Gilbert, D. J., Copeland, N. G., Jenkins, N.A., and Nathans, J. (1996)) and among ESTs obtained from human retinacDNA clones by the I.M.A.G.E. consortium (Lennon et al., 1996). ABCR isclosely related to the previously described mouse and human ABC1 andABC2 genes (Luciani et al., 1994; Allikmets et al., 1995). To determinewhether ABCR might cause a disease, the gene was mapped with a wholegenome radiation hybrid panel (GeneBridge 4; Research Genetics,Huntsville, Ala.). ABCR mapped to the human chromosome 1p13-p21 region,close to rnicrosatellite markers D1S236 and D1S188. To define furtherthe location of the gene, PCR primers, 3′UTR-For5′ATCCTCTGACTCAGCAATCACA, SEQ ID NO: 7, and 3′UTR-Rev5′TTGCAATTACAAATGCAATGG, SEQ ID NO: 8, from the putative 3′ untranslatedregion were used to screen YACs from the previously described contigbetween these anonymous markers (Anderson et al., 1995). At least 12YACs contain the 3′ end of the ABCR gene, including 924_e_(—)9,759_d_(—)7, 775_c_(—)2, 782_b_(—)4, 982_g_(—)5, 775_b_(—)2, 765_a_(—)3,751_f_(—)2, 848_e_(—)3, 943_h_(—)8, 934_g_(—)7, and 944_b_(—)12 (FIG.1). These YACs delineate a region containing the STGD gene betweenmarkers D1S3361 and D1S236 (Anderson et al., 1995).

Expression of the ABCR Gene

[0076] Additional support suggesting that ABCR is a candidate STGD genecame from expression studies and inspection of the EST databases.

[0077] Searches of the dbEST (Boguski et al., 1993) database wereperformed with BLAST on the NCBI file server (Altschul et al., 1990).Amino acid alignments were generated with PILEUP (Feng and Doolittle,1987). Sequences were analyzed with programs of the Genetics ComputerGroup package (Devereaux et al., 1984) on a VAX computer.

[0078] Clones corresponding to the mouse ortholog of the human ABCR genewere isolated from the mouse retina cDNA library and end-sequenced. Thechromosomal location of the mouse ABCR gene was determined on TheJackson Laboratory (Bar Harbor, Me.) interspecific backcross mappingpanel (C57BL/6JEi X SPRET/Ei)F1 X SPRET/Ei (Rowe et al., 1994) known asJackson BSS. Mapping was performed by SSCP analysis with the primersMABCR1F 5′ATC CAT ACC CTT CCC ACT CC, SEQ ID NO: 9, and MABCR1R 5′ GCAGCA GAA GAT AAG CAC ACC, SEQ ID NO. 10. The allele pattern of the Abcrwas compared to the 250 other loci mapped previously in the Jackson BSScross (http://www.jax.org).

[0079] DNA fragments used as probes were purified on a 1% low-meltingtemperature agarose gel. The probe sequences are set forth within thegenomic sequence of SEQ ID NO: 1 and FIG. 3. DNA was labeled directly inagarose with the Random Primed DNA Labeling Kit (Boehringer Mannheim,Indianapolis, Ind.) and hybridized to multiple tissue Northern blot anda Master blot (Clontech, Palo Alto, Calif.), according to themanufacturer's instructions. Each blot contained 2 μg of poly A+ RNAfrom various human tissues. Total RNA was isolated from adult rattissues using the guanidinium thiocyanate method (Chomczynski andSaachi, 1987) and resolved by agarose gel electrophoresis in thepresence of formaldehyde (Sambrook et al., 1989). Hybridization with themouse ABCR probe was performed in 50% formamide, 5×SSC at 42° C., andfilters were washed in 0.1×SSC at 68° C.

[0080] Hybridization of a 3′ ABCR cDNA probe to a multiple tissueNorthern blot and a MasterBlot (Clontech, Palo Alto, Calif.) indicatedthat the gene was not expressed detectably in any of the 50 non-retinalfetal and adult tissues examined, consistent with the observation thatall 12 of the ABCR clones in the EST database originated from retinalcDNA libraries. Furthermore, screening cDNA libraries from bothdeveloping mouse eye and adult human retina with ABCR probes revealed anestimated at 0.1%-1% frequency of ABCR clones of all cDNA clones in thelibrary. Hybridization of the ABCR probe to a Northern blot containingtotal RNA from rat retina and other tissues showed that the expressionof this gene is uniquely retina-specific (FIG. 2). The transcript sizeis estimated to be 8 kb.

Sequence and Exon/Intron Structure of the ABCR cDNA

[0081] Several ESTs that were derived from retina cDNA libraries and hadhigh similarity to the mouse Abc1 gene were used to facilitate theassembly of most of the ABCR cDNA sequence. Retina cDNA clones werelinked by RT-PCR, and repetitive screening of a human retina cDNAlibrary with 3′ and 5′ PCR probes together with 5′ RACE were used tocharacterize the terminal sequences of the gene.

[0082] cDNA clones containing ABCR sequences were obtained from a humanretina cDNA library (Nathans et al., 1986) and sequenced fully. Primerswere designed from the sequences of cDNA clones from 5′ and 3′ regionsof the gene and used to link the identified cDNA clones by RT-PCR withretina QUICK-Clone cDNA (Clontech, Palo Alto, Calif.) as a template. PCRproducts were cloned into pGEM®-T vector (Promega, Madison, Wis.). MouseABCR CDNA clones were obtained from screening a developing mouse eyecDNA library (H. Sun, A. Lanahan, and J. Nathans, unpublished). ThepGEM®-T Vector is prepared by cutting pGEM®-5Zf(+) DNA with EcoR V andadding to a 3′ terminal thymidine to both ends. These single 3′-Toverhangs at the insertion site greatly improve the efficiency ofligation of PCR products because of the nontemplate-dependent additionof a single deoxyadenosine (A) to the 3′-ends of PCR products by manythermostable poly erases. The pGEM®-5Zf(+) Vector contains the origin ofreplication of the filamentous phage f1 and can be used to producessDNA. The plasmid also contains T7 and SP6 RNA polymerase promotersflanking a multiple cloning region within the α-peptide coding regionfor the enzyme β-galactosidase. Insertional inactivation of theα-peptide allows recombinant clones to be identified directly by colorscreening on indicator plates. cDNA clones from various regions of theABCR gene were used as probes to screen a human genomic library inLambda FIX II (#946203, Stratagene, LaJolla, Calif.). Overlapping phageclones were mapped by EcoRI and BamHI digestion. A total of 6.9 kb ofthe ABCR sequence was assembled, (FIG. 3) resulting in a 6540 bp (2180amino acid) open reading frame.

[0083] Screening of a bacteriophage lambda human genomic library withcDNA probes yielded a contig that spans approximately 100 kb andcontains the majority of the ABCR coding region. The exon/intronstructure of all fifty one exons of the gene were characterized bydirect sequencing of genomic and cDNA clones. Intron sizes wereestimated from the sizes of PCR products using primers from adjacentexons with genomic phage clones as templates.

[0084] Primers for the cDNA sequences of the ABCR were designed with thePRIMER program (Lincoln et al., 1991). Both ABCR cDNA clones and genomicclones became templates for sequencing. Sequencing was performed withthe Taq Dyedeoxy Terminator Cycle Sequencing kit (Applied Biosystems,Foster City, Calif.), according to the manufacturer's instructions.Sequencing reactions were resolved on an ABI 373A automated sequencer.Positions of introns were determined by comparison between genomic andcDNA sequences. Primers for amplification of individual exons weredesigned from adjacent intron sequences 20-50 bp from the splice siteand are set forth in Table 1. TABLE 1 Exon/intron Primers for ABCRPRIMER SEQUENCE SEQ ID NO ABCR.EXON1:F ACCCTCTGCTAAGCTCAGAG 12ABCR.EXON1:R ACCCCACACTTCCAACCTG 13 ABCR.EXON2:F AAGTCCTACTGCACACATGG 14ABCR.EXON2:R ACACTCCCACCCCAAGATC 15 ABCR.EXON3:F TTCCCAAAAAGGCCAACTC 16ABCR.EXON3:R CACGCACGTGTGCATTCAG 17 ABCR.EXON4:F GCTATTTCCTTATTAATGAGGC18 ABCR.EXON4:R CCAACTCTCCCTGTTCTTTC 19 ABCR.EXON5:FTGTTTTCCAATCGACTCTGGC 20 ABCR.EXON5:R TTCTTGCCTTTCTCAGGCTGG 21ABCR.EXON6:F GTATTCCCAGGTTCTGTGG 22 ABCR.EXON6:R TACCCCAGGAATCACCTTG 23ABCR.EXON7:F AGCATATAGGAGATCAGACTG 24 ABCR.EXON7:R TGACATAAGTGGGGTAAATGG25 ABCR.EXON8:F GAGCATTGGCCTCACAGCAG 26 ABCR.EXON8:R CCCCAGGTTTGTTTCACC27 ABCR.EXON9:F AGACATGTGATGTGGATACAC 28 ABCR.EXON9:RGTGGGAGGTCCAGGGTACAC 29 ABCR.EXON10:F AGGGGCAGAAAAGACACAC 30ABCR.EXON10:R TAGCGATTAACTCTTTCCTGG 31 ABCR.EXON11:F CTCTTCAGGGAGCCTTAGC32 ABCR.EXON11:R TTCAAGACCACTTGACTTGC 33 ABCR.EXON12:FTGGGACAGCAGCCTTATC 34 ABCR.EXON12:R CCAAATGTAATTTCCCACTGAC 35ABCR.EXON13:F AATGAGTTCCGAGTCACCCTG 36 ABCR.EXON13:R CCCATTCGCGTGTCATGG37 ABCR.EXON14:F TCCATCTGGGCTTTGTTCTC 38 ABCR.EXON14:RAATCCAGGCACATGAACAGG 39 ABCR.EXON15:F AGGCTGGTGGGAGAGAGC 40ABCR.EXON15:R AGTGGACCCCCTCAGAGG 41 ABCR.EXON16:F CTGTTGCATTGGATAAAAGGC42 ABCR.EXON16:R GATGAATGGAGAGGGCTGG 43 ABCR.EXON17:FCTGCGGTAAGGTAGGATAGGG 44 ABCR.EXON17:R CACACCGTTTACATAGAGGGC 45ABCR.EXON18:F CCTCTCCCCTCCTTTCCTG 46 ABCR.EXONI8:R GTCAGTTTCCGTAGGCTTC47 ABCR.EXON19:F TGGGGCCATGTAATTAGGC 48 ABCR.EXON19:RTGGGAAAGAGTAGACAGCCG 49 ABCR.EXON20:F ACTGAACCTGGTGTGGGG 50ABCR.EXON20:R TATCTCTGCCTGTGCCCAG 51 ABCR.EXON21:F GTAAGATCAGCTGCTGGAAG52 ABCR.EXON21:R GAAGCTCTCCTGCACCAAGC 53 ABCR.EXON22:FAGGTACCCCCACAATGCC 54 ABCR.EXON22:R TCATTGTGGTTCCAGTACTCAG 55ABCR.EXON23:F TTTTTGCAACTATATAGCCAGG 56 ABCR.EXON23:RAGCCTGTGTGAGTAGCCATG 57 ABCR.EXON24:F GCATCAGGGCGAGGCTGTC 58ABCR.EXON24:R CCCAGCAATACTGGGAGATG 59 ABCR.EXON25:F GGTAACCTCACAGTCTTCC60 ABCR.EXON25:R GGGAACGATGGCTTTTTGC 61 ABCR.EXON26:FTCCCATTATGAAGCAATACC 62 ABCR.EXON26:R CCTTAGACTTTCGAGATGG 63ABCR.EXON27:F GCTACCAGCCTGGTATTTCATTG 64 ABCR.EXON27:RGTTATAACCCATGCCTGAAG 65 ABCR.EXON28:F TGCACGCGCACGTGTGAC 66ABCR.EXON28:R TGAAGGTCCCAGTGAAGTGGG 67 ABCR.EXON29:FCAGCAGCTATCCAGTAAAGG 68 ABCR.EXON29:R AACGCCTGCCATCTTGAAC 69ABCR.EXON30:F GTTGGGCACAATTCTTATGC 70 ABCR.EXON30:R GTTGTTTGGAGGTCAGGTAC71 ABCR.EXON31:F AACATCACCCAGCTGTTCCAG 72 ABCR.EXON31:RACTCAGGAGATACCAGGGAC 73 ABCR.EXON32:F GGAAGACAACAAGCAGTTTCAC 74ABCR.EXON32:R ATCTACTGCCCTGATCATAC 75 ABCR.EXON33:FAAGACTGAGACTTCAGTCTTC 76 ABCR.EXON33:R GGTGTGCCTTTTAAAAGTGTGC 77ABCR.EXON34:F TTCATGTTTCCCTACAAAACCC 78 ABCR.EXON34:RCATGAGAGTTTCTCATTCATGG 79 ABCR.EXON35:F TGTTTACATGGTTTTTAGGGCC 80ABCR.EXON35:R TTCAGCAGGAGGAGGGATG 81 ABCR.EXON36:FCCTTTCCTTCACTGATTTCTGC 82 ABCR.EXON36:R AATCAGCACTTCGCGGTG 83ABCR.EXON37:F TGTAAGGCCTTCCCAAAGC 84 ABCR.EXON37:R TGGTCCTTCAGCGCACACAC85 ABCR.EXON38:F CATTTTGCAGAGCTGGCAGC 86 ABCR.EXON38:RCTTCTGTCAGGAGATGATCC 87 ABCR.EXON39:F GGAGTGCATTATATCCAGACG 88ABCR.EXON39:R CCTGGCTCTGCTTGACCAAC 89 ABCR.EXON40:F TGCTGTCCTGTGAGAGCATC90 ABCR.EXON40:R GTAACCCTCCCAGCTTTGG 91 ABCR.EXON41:FCAGTTCCCACATAAGGCCTG 92 ABCR.EXON41:R CAGTTCTGGATGCCCTGAG 93ABCR.EXON42:F GAAGAGAGGTCCCATGGAAAGG 94 ABCR.EXON42:RGCTTGCATAAGCATATCAATTG 95 ABCR.EXON43:F CTCCTAAACCATCCTTTGCTC 96ABCR.EXON43:R AGGCAGGCACAAGAGCTG 97 ABCR.EXON44:F CTTACCCTGGGGCCTGAC 98ABCR.EXON44:R CTCAGAGCCACCCTACTATAG 99 ABCR.EXON45:FGAAGCTTCTCCAGCCCTAGC 100 ABCR.EXON45:R TGCACTCTCATGAAACAGGC 101ABCR.EXON46:F GTTTGGGGTGTTTGCTTGTC 102 ABCR.EXON46:RACCTCTTTCCCCAACCCAGAG 103 ABCR.EXON47:F GAAGCAGTAATCAGAAGGGC 104ABCR.EXON47:R GCCTCACATTCTTCCATGCTG 105 ABCR.EXON48:FTCACATCCCACAGGCAAGAG 106 ABCR.EXON48:R TTCCAAGTGTCAATGGAGAAC 107ABCR.EXON49:F ATTACCTTAGGCCCAACCAC 108 ABCR.EXON49:R ACACTGGGTGTTCTGGACC109 ABCR.EXON50:F GTGTAGGGTGGTGTTTTCC 110 ABCR.EXON50:RAAGCCCAGTGAACCAGCTGG 111 ABCR.EXON51:F TCAGCTGAGTGCCCTTCAG 112ABCR.EXON51:R AGGTGAGCAAGTCAGTTTCGG 113

[0085] In Table 1, “F” indicates forward, i.e., 5′ to 3′, “R” indicatesreverse, i.e., 3′ to 5′. PCR conditions were 95° C. for 8 minutes; 5cycles at 62° C. for 20 seconds, 72° C. for 30 seconds; 35 cycles at 60°C. for 20 seconds, 72° C. for 30 seconds; 72° C. for 5 minutes (exceptthat ^(a) was performed at 94°C. for 5 minutes); 5 cycles at 94° C. for40 seconds; 60° C. for 30 seconds; 72° C. for 20 seconds; 35 cycles at94 C. for 40 seconds; 56° C. for 30 seconds; 62° C. for 20 seconds, and72° C. for 5 minutes.

[0086] Amplification of exons was performed with AmpliTaq Goldpolymerase in a 25 μl volume in 1×PCR buffer supplied by themanufacturer (Perkin Elmer, Foster City, Calif.). Samples were heated to95° C. for 10 minutes and amplified for 35-40 cycles at 96° C. for 20seconds; 58° C. for 30 seconds; and 72° C. for 30 seconds. PCR productswere analyzed on 1-1.5% agarose gels and in some cases digested with anappropriate restriction enzymes to verify their sequence. Primersequences and specific reaction conditions are set forth in Table 1. Thesequence of the ABCR cDNA has been deposited with GenBank underaccession #U88667.

Homology to ABC Superfamily Members

[0087] A BLAST search revealed that ABCR is most closely related to thepreviously characterized mouse Abc1 and Abc2 genes (Luciani et al.,1994) and to another human gene (ABCC) which maps to chromosome 16p13.3(Klugbauer and Hofinann, 1996). These genes, together with ABCR and agene from C. elegans (GenBank #Z29117), form a subfamily of genesspecific to multicellular organisms and not represented in yeast(Michaelis and Berkower, 1995; Allikmets et al., 1996). Alignment of theCDNA sequence of ABCR with the Abc1, Abc2, and ABCC genes revealed, asexpected, the highest degree of homology within the ATP-bindingcassettes. The predicted amino acid identity of the ABCR gene to mouseAbc1 was 70% within the ATP-binding domains; even within hydrophobicmembrane-spanning segments, homology ranged between 55 and 85% (FIG. 4).The putative ABCR initiator methionine shown in FIGS. 3 and 4corresponds to a methionine codon at the 5′ end of Abc1 (Luciani et al.,1994).

[0088] ABCR shows the composition of a typical full-length ABCtransporter that consists of two transmembrane domains (TM), each withsix membrane spanning hydrophobic segments, as predicted by a hydropathyplot (data not shown), and two highly conserved ATP-binding domains(FIGS. 3 and 4). In addition, the HH1 hydrophobic domain, locatedbetween the first ATP and second TM domain and specific to thissubfamily (Luciani et al., 1994), showed a predicted 57% amino acididentity (24 of 42 amino acids) with the mouse Abc1 gene.

[0089] To characterize the mouse ortholog of ABCR, cDNA clones from adeveloping mouse eye library were isolated. A partial sequence of themouse cDNA was utilized to design PCR primers to map the mouse Abcr genein an interspecific backcross mapping panel (Jackson BSS). The allelepattern of Abcr was compared to 2450 other loci mapped previously in theJackson BSS cross; linkage was found to the distal end of chromosome 3(FIG. 5). No recombinants were observed between Abcr and D13Mit13. Thisregion of the mouse genome is syntenic with human chromosome 1p13-p21.Thus far, no eye disease phenotype has been mapped to this region ofmouse chromosome 3.

Compound Heterozygous and Homozygous Mutations in STGD Patients

[0090] One hundred forty-five North American and three Saudi Arabianfamilies with STGD/FFM were examined. Among these, at least four wereconsanguineous families in which the parents were first cousins. Entrycriteria for the characterization of the clinical and angiographicdiagnosis of Stargardt disease, ascertainment of the families, andmethodology for their collection, including the consanguineous familiesfrom Saudi Arabia, were as provided in Anderson et al., 1995; andAnderson, 1996.

[0091] Mutational analysis of the ABCR gene was pursued in the aboveidentified one hundred forty-eight STGD families previously ascertainedby strict definitional criteria and shown to be linked to chromosome 1p(Anderson et al., 1995; Anderson, 1996). To date, all 51 exons have beenused for mutation analysis.

[0092] Mutations were detected by a combined SSCP (Orita et al., 1989)and heteroduplex analysis (White et al., 1992) under optimizedconditions (Glav{haeck over (c)}and Dean, 1993). Genomic DNA samples (50ng) were amplified with AmpliTaq Gold polymerase in 1×PCR buffersupplied by the manufacturer (Perkin Elmer, Foster City, Calif.)containing [α-³²p] dCTP. Samples were heated to 95° C. for 10 minutesand amplified for 35-40 cycles at 96° C. for 20 seconds; 58° C. for 30seconds; and 72° C. for 30 seconds. Products were diluted in 1:3 stopsolution, denatured at 95° C. for 5 minutes, chilled in ice for 5minutes, and loaded on gels. Gel formulations include 6% acrylamide:Bis(2.6% cross-linking), 10% glycerol at room temperature, 12W; and 10%acrylamide:Bis (1.5% cross-linking), at 4° C., 70W. Gels were run for2-16 hours (3000 Vh/100 bp), dried, and exposed to X-ray film for 2-12hours. Some exons were analyzed by SSCP with MDE acrylamide (FMCBioproducts, Rockland, Me.) with and without 10% glycerol for 18 hours,4 watts at room temperature with α-P³²-dCTP labeled DNA. Heteroduplexeswere identified from the double-stranded DNA at the bottom of the gels,and SSCPs were identified from the single-stranded region. Samplesshowing variation were compared with other family members to assesssegregation of the alleles and with at least 40 unrelated controlsamples, from either Caucasian or Saudi Arabian populations, todistinguish mutations from polymorphisms unrelated to STGD. PCR productswith SSCP or heteroduplex variants were obtained in a 25 μl volume,separated on a 1% agarose gel, and isolated by a DNA purification kit(PGC Scientific, Frederick, Md.). Sequencing was performed on an ABIsequencer with both dye primer and dye terminator chemistry.

[0093] Some mutations were identified with a heteroduplex analysisprotocol (Roa et al., 1993). Equimolar amounts of control and patientPCR products were mixed in 0.2 ml tubes. Two volumes of PCR product froma normal individual served as a negative control, and MPZ exon 3 frompatient BAB731 as a positive control (Roa et al., 1996). Samples weredenatured at 95 ° C. for 2 minutes and cooled to 35° C. at a rate of 1°C./minute. Samples were loaded onto 1.0 mm thick, 40 cm MDE gels (FMCBioproducts, Rockland, Me.), electrophoresed at 600-800 V for 15-20hours, and visualized with ethidium bromide. Samples showing a variantband were reamplified with biotinylated forward and reverse primers andimmobilized on streptavidin-conjugated beads (Warner et al. 1996). Theresulting single strands were sequenced by the dideoxy-sequencing methodwith Sequenase 2.0 (Amersham, Arlington Heights, Ill.).

[0094] A total of seventy five mutations were identified, the majorityrepresenting missense mutations in conserved amino acid positions.However, several insertions and deletions representing frameshifts werealso found (Table 2). The sequence of two mutations are shown in FIG. 6Aand 6B. Two missense alterations (D847H, R943Q) were found in at leastone control individual, suggesting that they are neutral polymorphisms.The remaining mutations were found in patients having maculardegeneration and were not found in at least 220 unrelated normalcontrols (440 chromosomes), consistent with the interpretation thatthese alterations represent disease-causing mutations, notpolymorphisms. One of the mutations, 5892+1 G→T, occurs in family AR144in which one of the affected children is recombinant for the flankingmarker D1S236 (Anderson et al., 1995). This mutation, however, ispresent in the father as well as in both affected children. Therefore,the ABCR gene is non-recombinant with respect to the Stargardt diseaselocus.

[0095] The mutations are scattered throughout the coding sequence of theABCR gene (see Table 2 and FIG. 3), although clustering within theconserved regions of the ATP-binding domains is noticeable. Homozygousmutations were detected in three likely consanguineous families, twoSaudi Arabian and one North American (Anderson et al., 1995), in each ofwhich only the affected individuals inherited the identical diseaseallele (Table 2; FIG. 6C). Forty two compound heterozygous families wereidentified in which the two disease alleles were transmitted fromdifferent parents to only the affected offspring (Table 2). TABLE 2Mutations in the ABCR gene in STGD Families Nucleotide Amino Acid#Families Exon 0223T->G C75G 1 3 0634C->T R212C 1 6 0664del13 fs 1 60746A->G D249G 1 6 1018T->G Y340D 2 8 1411G->A E471K 1 11 1569T->G D523E1 12 1715G->A R572Q 2 12 1715G->C R572P 1 12 1804C->T R602W 1 131822T->A F608I 1 13 1917C->A Y639X 1 13 2453G->A G818E 1 16 2461T->AW821R 1 16 2536G->C D846H 1 16 2588G->C G863A 11 17 2791G->A V931M 1 192827C->T R943W 1 19 2884delC fs 1 19 2894A->G N965S 3 19 3083C->T A1028V14 21 3211delGT fs 1 22 3212C->T S1071L 1 22 3215T->C V1072A 1 223259G->A E1087K 1 22 3322C->T R1108C 6 22 3364G->A E1122K 1 23 3385G->TR1129C 1 23 3386G->T R1129L 1 23 3602T->G L1201R 1 24 3610G->A D1204N 125 4139C->T P1380L 2 28 4195G->A E1399K 1 28 4222T->C W1408R 3 284232insTATG fs 1 28 4253+5G->T splice 1 28 4297G->A V1433I 1 29 4316G->AG1439D 1 29 4319T->C F1440S 1 29 4346G->A W1449X 1 29 4462T->C C1488R 130 4469G->A C1490Y 1 31 4577C->T T1526M 6 32 4594G->A D1532N 2 324947delC fs 1 36 5041del15 VVAIC1681del 1 37 5196+2T->C splice 1 375281del9 PAL1761del 1 38 5459G->C R1820P 1 39 5512C->T H1838Y 1 405527C->T R1843W 1 40 5585+1G->A splice 1 41 5657G->A G1886E 1 415693G->A R1898H 4 41 5714+5G->A splice 8 41 5882G->A G1961E 16 435898+1G->A splice 3 43 5908C->T L1970F 1 44 5929G->A G1977S 1 446005+1G->T splice 1 44 6079C->T L2027F 11 45 6088C->T R2030X 1 456089G->A R2030Q 1 45 6112C->T R2038W 1 45 6148G->C V2050L 2 46 6166A->TK2056X 1 46 6229C->T R2077W 1 46 6286G->A E2096K 1 47 6316C->T R2106C 147 6391G->A E2131K 1 48 6415C->T R2139W 1 48 6445C->T R2149X 1 486543del36 1181del12 1 49 6709delG fs 1 49

[0096] Mutations are named according to standard nomenclature. Thecolumn headed “Exon” denotes which of the 51 exons of ABCR contain themutation. The column headed “# Families” denotes the number of Stargardtfamilies which displayed the mutation. The column headed “Nucleotide”gives the base number starting from the A in the initiator ATG, followedby the wild type sequence and an arrow indicating the base it is changedto; del indicates a deletion of selected bases at the given position inthe ABCR gene; ins indicates an insertion of selected bases at the givenposition; splice donor site mutations are indicated by the number of thelast base of the given exon, followed by a plus sign and the number ofbases into the intron where the mutation occurs. The column headed“Amino Acid” denotes the amino acid change a given mutation causes; fsindicates a frameshift mutation leading to a truncated protein; spliceindicates a splice donor site mutation; del indicates an in-framedeletion of the given amino acids.

[0097] Mutations are named according to standard nomenclature. Exonnumbering according to the nucleotide position starting from the A inthe initiator ATG.

In Situ Hybridization

[0098] STGD is characterized histologically by a massive accumulation ofa lipofuscin-like substance in the retinal pigment epithelium (RPE).This characteristic has led to the suggestion that STGD represents anRPE storage disorder (Blacharski et al., 1988). It was therefore ofinterest that ABCR transcripts were found to be abundant in the retina.To identify the site(s) of ABCR gene expression at higher resolution andto determine whether the gene is also expressed in the RPE, thedistribution of ABCR transcripts was visualized by in situ hybridizationto mouse, rat, bovine, and macaque ocular tissues.

[0099] In situ hybridization with digoxigenin-labeled riboprobes wasperformed as described by Schaeren-Wiemers and Gerfin-Moser, 1993. Formouse and rat, unfixed whole eyes were frozen and sectioned; macaqueretinas were obtained following cardiac perfusion with paraformaldehydeas described (Zhou et al., 1996). An extra incubation of 30 min in 1%Triton X-100, 1×PBS was applied to the fixed monkey retina sectionsimmediately after the acetylation step. The templates for probesynthesis were: (1) a 1.6 kb fragment encompassing the 3′ end of themouse Abcr coding region, (2) a full length cDNA clone encoding themouse blue cone pigment (Chiu et al., 1994), and (3) a macaque rhodopsincoding region segment encoding residues 133 to 254 (Nickells, R. W.,Burgoyne, C. F., Quigley, H. A., and Zack, D. J. (1995)).

[0100] This analysis showed that ABCR transcripts are presentexclusively within photoreceptor cells (FIG. 7). ABCR transcripts arelocalized principally to the rod inner segments, a distribution thatclosely matches that of rhodopsin gene transcripts. Interestingly, ABCRhybridization was not observed at detectable levels in conephotoreceptors, as judged by comparisons with the hybridization patternsobtained with a blue cone pigment probe (compare FIG. 7A and FIG. 7D,FIG. 7E with FIG. 7F and FIG. 7G with FIG. 7H). Because melanin granulesmight obscure a weak hybridization signal in the RPE of a pigmentedanimal, the distribution of ABCR transcripts was also examined in bothalbino rats and albino mice. In these experiments, the ABCRhybridization signal was seen in the photoreceptor inner segments andwas unequivocally absent from the RPE (FIG. 7E). Given that ABCRtranscripts in each of these mammals, including a primate, arephotoreceptor-specific, it is highly likely that the distribution ofABCR transcripts conforms to this pattern as well in the human retina.

[0101] The disclosures of each patent, patent application andpublication cited or described in this document are hereby incorporatedherein by reference, in their entirety.

[0102] Various modifications of the invention in addition to those shownand described herein will be apparent to those skilled in the art fromthe foregoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

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What is claimed is:
 1. An isolated nucleic acid sequence encodingretina-specific ATP binding cassette transporter.
 2. An isolated nucleicacid sequence selected from the group consisting of SEQ ID NO: 1, or afragment thereof having substantially the same activity.
 3. An isolatednucleic acid sequence selected from the group consisting of SEQ ID NOS:2 or 5, or a fragment thereof having substantially the same activity. 4.An isolated amino acid sequence selected from the group consisting ofSEQ ID NO: 3 or 6, or a fragment thereof having substantially the sameactivity.
 5. An isolated amino acid sequence of FIG. 3, or a fragmentthereof having substantially the same activity.
 6. A vector comprising anucleic acid sequence encoding retina-specific ATP binding cassettetransporter.
 7. A vector comprising a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, or a fragment thereof havingsubstantially the same activity.
 8. A vector comprising a nucleic acidsequence selected from the group consisting of SEQ ID NOS: 2 or 5, or afragment thereof having substantially the same activity.
 9. A vectorcomprising a nucleic acid sequence encoding an amino acid sequenceselected from the group consisting of SEQ ID NOS: 3 or
 6. 10. A vectorcomprising a nucleic acid sequence encoding the amino acid sequence ofFIG.
 3. 11. A host cell capable of expressing a nucleic acid sequenceencoding a retina-specific ATP binding cassette transporter.
 12. A hostcell capable of expressing a nucleic acid sequence of SEQ ID NO:
 1. 13.A host cell capable of expressing a nucleic acid sequence selected fromthe group consisting of SEQ ID NOS: 2 or
 5. 14. A host cell capable ofexpressing a nucleic acid sequence encoding an amino acid sequenceselected from the group consisting of SEQ ID NOS: 3 or
 6. 15. A hostcell capable of expressing a nucleic acid sequence encoding the aminoacid sequence of FIG.
 3. 16. A cell culture capable of expressing aretina-specific ATP binding cassette transporter.
 17. A cell culturecapable of expressing a nucleic acid sequence of SEQ ID NO:
 1. 18. Acell culture capable of expressing a nucleic acid sequence selected fromthe group consisting of SEQ ID NOS: 2 or
 5. 19. A cell culture capableof expressing a nucleic acid sequence encoding an amino acid sequenceselected from the group consisting of SEQ ID NOS: 3 or
 6. 20. A cellculture of claim 19 obtained by transforming a cell with an expressionvector comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NOS: 2 or
 5. 21. A cell culture capable ofexpressing a nucleic acid sequence encoding an amino acid sequenceselected from the group consisting of SEQ ID NOS: 3 or
 6. 22. A proteinpreparation comprising an amino acid sequence for retina-specific ATPbinding cassette transporter.
 23. A protein preparation comprising anamino acid sequence encoded by a sequence of SEQ ID NO:
 1. 24. A proteinpreparation comprising an amino acid sequence encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NOS: 2 or
 5. 25. Aprotein preparation comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 3 or
 6. 26. A protein preparationcomprising an amino acid sequence of FIG.
 3. 27. A compositioncomprising an effective amount of a sequence selected from the groupconsisting of SEQ ID NOS: 2 or 5 or a fragment thereof havingsubstantially similar activity, and a pharmaceutically acceptablecarrier.
 28. A composition comprising an effective amount of anantisense sequence to a sequence selected from the group consisting ofSEQ ID NOS: 2 or 5 or a fragment thereof which fragment hassubstantially similar activity, and a pharmaceutically acceptablecarrier.
 29. A method of screening for an agent that altersretina-specific ATP binding cassette transporter comprising combiningpurified retina-specific ATP binding cassette transporter and at leastone agent suspected of altering retina-specific ATP binding cassettetransporter and observing an alteration in said purified retina-specificATP binding cassette transporter.
 30. The method of claim 29 whereinsaid alteration is activation of said purified retina-specific ATPbinding cassette transporter observed by a inhibition of acharacteristic associated with macular degeneration selected from thegroup consisting of inhibition of central visual impairment, inhibitionof progressive bilateral atrophy of the macular retinal pigmentepithelium, inhibition of progressive bilateral atrophy of theneuroepithelium, inhibition of macula flecks, inhibition of midretinalperiphery flecks, and inhibition of retina-specific ATP binding cassettetransporter transcripts in photoreceptor cells.
 31. The method of claim30 wherein said macular degeneration is selected from the groupconsisting of Stargardt Disease, Fundus Flavimaculatus, and age-relatedmacular degeneration.
 32. A method of claim 29 wherein said alterationis ari inhibition of said purified retina-specific ATP binding cassettetransporter observed by a characteristic associated with maculardegeneration selected from the group consisting of central visualimpairment, bilateral atrophy of the macular retinal pigment epithelium,bilateral atrophy of the neuroepithelium, macula flecks, midretinalperiphery flecks, and retina-specific ATP binding cassette transportertranscripts in photoreceptor cells.
 33. A method of screening for anagent that inhibits macular degeneration comprising combining purifiedretina-specific ATP binding cassette transporter from a patientsuspected of having macular degeneration and at least one agentsuspected of activating retina-specific ATP binding cassette transporterand observing an activation in said purified retina-specific ATP bindingcassette transporter.
 34. A method of screening for an agent thatactivates macular degeneration comprising combining a purified wild-typeretina-specific ATP binding cassette transporter and at least one agentsuspected of activating macular degeneration and observing an inhibitionin said purified wild-type retina-specific ATP binding cassettetransporter.
 35. A transgenic non-human mammal comprising a recombinantsequence encoding a retina-specific ATP binding cassette transporterintroduced into said mammal, or an ancestor of said mammal.
 36. Themammal of claim 35 wherein said sequence encoding said retina-specificATP binding cassette transporter is selected from the group consistingof SEQ ID NOS: 1, 2, and
 5. 37. A transgenic non-human mammal comprisinga suppressed retina-specific ATP binding cassette transporter gene. 38.A transgenic non-human mammal comprising a recombinant wild-typesequence encoding retina-specific ATP binding cassette transporter. 39.The transgenic non-human mammal of claim 35 wherein said retina-specificATP binding cassette transporter sequence is selected from the groupconsisting of SEQ ID NOS: 3 and
 6. 40. A diagnostic kit for detectingmacular degeneration comprising in one or more containers a pair ofprimers, wherein one primer within said pair is complementary to aregion of the retina-specific ATP binding cassette receptor, a probespecific to the amplified product, and a means for visualizing amplifiedDNA, and optionally including one or more size markers, and positive andnegative controls.
 41. The diagnostic kit of claim 40 wherein saidprimer is selected from the group consisting of SEQ ID NOS: 12-113. 42.The diagnostic kit of claim 40 wherein said primer is complementary to aregion flanking an exon of retina-specific ATP binding cassette receptorgenomic DNA sequence.
 43. The diagnostic kit of claim 40 wherein saidmeans for visualizing amplified DNA is selected from the groupconsisting of fluorescent stain, ³²p, and biotin.
 44. A method ofdetecting macular degeneration comprising: obtaining a sample comprisingpatient nucleic acids from a patient tissue sample; amplifyingretina-specific ATP binding cassette receptor specific nucleic acidsfrom said patient nucleic acids to produce a test fragment; obtaining asample comprising control nucleic acids from a control tissue sample;amplifying control nucleic acids encoding wild-type retina-specific ATPbinding cassette receptor to produce a control fragment; comparing thetest fragment with the control fragment to detect the presence of asequence difference in the test fragrnent, wherein a difference in saidtest fragment indicates macular degeneration.
 45. The method of claim 44wherein a sequence difference is selected from the group consisting of amissense mutation, an intragenic deletion, intragenic insertion, asplice donor site mutation, and a frameshift.
 46. The method of claim 44wherein a sequence difference is a missense mutation.
 47. The method ofclaim 44 wherein said amplification step comprises performing thepolyrnerase chain reaction.
 48. The method of claim 47 wherein thepolymerase chain reaction comprises using a pair of primers, wherein oneprimer within said pair is selected from the group consisting of SEQ IDNOS: 12-113.
 49. The method of claim 44 wherein said tissue sample isselected from the group consisting of blood, skin, serum, saliva,sputum, mucus, bone marrow, urine, lymph, a tear, chorion, and amnioticfluid.
 50. The method of claim 44 wherein said sequence difference isselected from the group consisting of 0223T→G, 0634C→T, 0746A→G,1018T→G, 1411G→A, 1569T→G, 1715G→A, 1715G→C, 1804C→T, 1822T→A, 1917C→A,2453G→A, 2461T→A, 2536G→C, 2588G→C, 2791G→A, 2827C→T, 2894A→G, 3083C→T,3212C→T, 3215T→C, 3259G→A, 3322C→T, 3364G→A, 3385G→T, 3386G→T, 3602T→G,3610G→A, 4139C→T, 4195G→A, 4222T→C, 4297G→A, 4316G→A, 4319T→C, 4346G→A,4462T→C, 4469G→A, 4577C→T, 4594G→A, 5041del15, 5281del9, 5459G→C,5512C→T, 5527C→T, 5657G→A, 5693G→A, 5882G→A, 5908C→T, 5929G→A, 6079C→T,6088C→T, 6089G→A, 6112C→T, 6148G→C, 6166A→T, 6229C→T, 6286G→A, 6316C→T,6391G→A, 6415C→T, 6445C→T, and 6543del36.
 51. The method of claim 44further wherein said sequence difference results in an amino acidsequence difference selected from the group consisting of C75G, R212C,D249G, Y340D, E471K, D523E, R572Q, R572P, R602W, F6081, Y639X, G818E,W821R, D846H, G863A, V931M, R943W, N965S, A1028V, S1071L,V1072A, E1087K,R1108C, E1122K, R1129C, R1129L, L1201R, D1204N, P1380L, E1399K, W1408R,V1433I, G1439D, F1440S, W1449X, C1488R, C1490Y, T1526M, D1532N,VVAIC1681del, PAL1761del, R1820P, H1838Y, R1843W, G1886E, R1898H,G1961E, L1970F, G1977S, L2027F, R2030X, R2030Q, R2038W, V2050L, K2056X,R2077W, E2096K, R2106C, E2131K, R2139W, R2149X, 1181del12, 0664del13,2884delC, 4232insTATG, 4947delC, 6709del G, 4253+5G→T, 5196+2T→C,5585+1G→A, 5714+5G→A, 5898+1G→A, and 6005+1G→T.
 52. The method of claim44 wherein said sequence difference results in a frame shift in theamino acid sequence.
 53. The method of claim 44 wherein said sequencedifference results in a splice site in the amino acid sequence.
 54. Asequence of having a sequence of SEQ ID NOS: 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, or
 113. 55. A sequence encoding SEQID NO: 2 having a mutation selected from the group consisting of0223T→G, 0634C→T, 0746A→G, 1018T→G, 1411G→A, 1569T→G, 1715G→A,1715G→C→1804C→T, 1822T→A, 1917C→A, 2453G→A, 2461T→A, 2536G→C, 2588G→C,2791G→A, 2827C→T, 2894A→G, 3083C→T, 3212C→T, 3215T→C, 3259G→A, 3322C→T,3364G→A, 3385G→T, 3386G→T, 3602T→G, 3610G→A, 4139C→T, 4195G→A, 4222T→C,4297G→A, 4316G→A, 4319T→C, 4346G→A, 4462T→C, 4469G→A, 4577C→T, 4594G→A,5041del15, 5281del9, 5459G→C, 5512C→T, 5527C→T, 5657G→A, 5693G→A,5882G→A, 5908C→T, 5929G→A, 6079C→T, 6088C→T, 6089G→A, 6112C→T, 6148G→C,6166A→T, 6229C→T, 6286G→A, 6316C→T, 6391G→A, 6415C→T, 6445C→T, and6543del36.
 56. A sequence of claim 55 wherein said sequence differenceresults in a frame shift in the amino acid sequence.
 57. The method ofclaim 55 wherein said sequence difference results in a splice site inthe amino acid sequence.
 58. A sequence encoding SEQ ID NO: 3 having amutation selected from the group consisting of C75G, R212C, D249G,Y340D, E471K, D523E, R572Q, R572P, R602W, F6081, Y639X, G818E, W821R,D846H, G863A, V931M, R943W, N965S, A1028V, S1071L,V1072A, E1087K,R1108C, E1122K, R1129C, R1129L, L1201R, D1204N, P1380L, E1399K, W1408R,V14331, G1439D, F1440S, W1449X, C1488R, Ci490Y, T1526M, D1532N,VVAIC1681del, PAL1761del, R1820P, H1838Y, R1843W, G1886E, R1898H,G1961E, L1970F, G1977S, L2027F, R2030X, R2030Q, R2038W, V2050L, K2056X,R2077W, E2096K, R2106C, E2131K, R2139W, R2149X, 1181del12, 0664del 13,2884delC, 4232insTATG, 4947delC, 6709delG, 4253+5G→T, 5196+2T→C,5585+1→A, 5714+5G→A, 5898+1G→A, and 6005+1G→T.